The invention relates to a process for producing L-carnitine from crotonobetaine, from salts of crotonobetaine, other derivatives of crotonobetaine or the like.
It is known that L-carnitine, a ubiquitously occurring compound, plays an important role in metabolism, especially in transporting long-chain fatty acids through the inner mitochondrial membrane. Numerous clinical applications derive from the function of carnitine in the metabolism of eukaryotes, e.g., in the treatment of patients with carnitine deficiency syndromes, in the prevention and therapy of various heart diseases and in the treatment of hemodialysis patients. Further, L-carnitine is significant as a supplemental nutrient and also promotes, as an additive to fermentation media, the growth of yeasts and bacteria. The growing need for this biologically active L-carnitine enantiomer for these and other applications has led to a worldwide search for means of synthesizing this betaine in an optically pure form, since the chemically synthesized racemate cannot be used because it inhibits carnitine acetyl transferase and the carnitine carrier protein.
To isolate the L-isomer, up to now processes have been used that are based on splitting racemates by fractionated crystallization using optically active acids (e.g., U.S. Pat. No. 4,254,053, 1981), where D(+)-carnitine occurs as a waste product.
This problem can be overcome by various biological processes, starting with inexpensive achiral precursors (Adv. Biochem. Eng. Biotechnol., 1993, 50, 21-44) Of particular interest is stereospecific hydration of trans-crotonobetaine into L-carnitine using strains of the genera Escherlchia (ED 0121444, 1984; DD 221 905, 1987; EP 0320460, 1989) or Proteus (Agric. Biol. Chem., 1988, 52, 2415-2421; U.S. Pat. No. 5,300,430, 1994) The advantage of this method lies in the fact that this achiral precursor can also be obtained by chemical dehydration of the waste product D-carnitine.
The numerous processes described in the literature with immobilized microorganisms in a continuously operating reactor system have the advantage that
pure reaction media can be used, thus facilitating the extraction and purification process,
by using higher concentrations of the biocatalyst in the reaction medium, higher productivities are achieved while the possibility of contamination is reduced,
there is reduced sensitivity to inhibitors or a nutrient deficiency,
a higher stability of the biocatalyst is achieved.
The advantages mentioned can also be applied to a commercially used process.
A continuously operating reactor in which microorganisms are retained by micro- or ultrafiltration membranes is an immobilization process which, besides the above-mentioned advantage, also entails lower costs for the immobilization while making it possible to have a very slight upscaling.
Consequently the object of the invention is a process for producing L-carnitine from crotonobetaine, crotonobetaine salts or other crotonobetaine derivatives in a continuous reactor with free or immobilized cells, growing or resting Escherlchla coil 044K74 (DSM 8828) cells, that are retained by micro- or ultrafiltration membranes in a flat membrane or hollow fiber module.
E. coil is kept in the reactor mentioned at temperatures between 20 and 40xc2x0 C., pH values between pH 6.0 8.0 and under anaerobic conditions that are necessary for the induction of the enzyme that metabolizes carnitine.
A minimal or complex medium is used as the reaction medium. In both cases, crotonobetaine, crotonobetaine salts or other crotonobetaine derivatives are added in concentrations between 25 mmol and 1M. The minimal medium contains varying concentrations of caseine hydrolyzate and salts (NH4)2504, KH2PO4, K2HPO4, MgSO4x7H20, MnSO4x4H20, FeSO4x7H20, while the complex medium contains varying concentrations of pancreatic peptone and NaCl. To improve the growth of E. coil, glycerine, glucose, ribose, saccharose or lactose are added. Also added to the medium are inhibitors that prevent the transformation of crotonobetaine into y-butyrobetaine (fumarate, glucose or nitrate) and inductors of carnitine-metabolizing enzymes such as D-, L-, DL-carnitine, their salts and derivatives or crotonobetaine, its salts or derivatives.
The course of the reaction in the continuous cell-recycle reactor used here can be divided into two stages. The one stage consists of a reactor tank in which cells of E. coil, together with the reaction medium, convert most of the crotonobetaine into L-carnitine. This reactor tank has monitoring elements for pH value, temperature and stirring speed and for the monitoring and correction of oxygen concentration. The feed of the reaction medium into the reactor is performed with a dosing pump. When necessary, excess medium must be removed from the reactor tank. The second stage consists of an external recycling loop that is connected to the reactor tank and conveys the contents of the reactor through a filter unit by means of a pump. While the filtrate is being collected, to isolate L-carnitine from it as the reaction product, the residue from the filtration is fed again to the reactor. For filtering the cell suspension, commercial filter systems of varying provenance can be used as long as they have a pore size below the cell size of E. coil. The speed of the recycling pump remains unchanged to achieve the best possible filtration rates and to minimize the formation of a polarization membrane during the filtration process. Filtering may be performed using commercial cross current filtration or hollow-fiber modules consisting of ultra-or microfiltration membranes composed of cellulose, polysulphone or polysulphonated polysulphone with a retention limit of 300 kDa or 0.211 xcexc. The continuous cell-recycle reactor may be operated at different levels of dilution adjusted by dosing and filtration pumps, and at different agitation speeds and different biomass concentrations. Rate of discharge from the filtration pump is controlled by process control means.
The expression free E. coil cells indicates the state in which whole cells are suspended in the reaction medium without preventing a cell outflow through the exit solution. The expression immobilized cells describes the state in which whole cells are bonded to soluble polymers or insoluble carriers, or are enclosed in membrane systems (in Methods in Enzymol. 1987, vol. 135, 3-30)
The concept growth conditions is defined as the situation in which whole cells use substrates and form products during their life cycle. Resting cells are understood as intact cells that are not growing and that show, under certain conditions, special metabolic functions (in xe2x80x9cBiotechnologyxe2x80x9d (Kieslich, K.; Eds. Rehm, N.J. and Reed, G.) Verlag Chemie, Weinheim, Germany. 1984, Vol. 6a, 5-30)
The process is described below with several embodiments: